Dissertations / Theses: 'Gene expression in bone cells'

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Kim, Michael S. "Gene Expression in Bone Cells." Thesis, Griffith University, 2006. http://hdl.handle.net/10072/366180.

Abstract:

Osteoclast formation is a complex process that is yet to be clearly defined. Osteoclasts differentiate from monocytic precursors to large multinuclear cells via the actions of two crucial cytokines: macrophage colony stimulating factor (M-CSF) and receptor activator of NFKB ligand (RANKL). These two cytokines bind to the osteoclast precursor cells, activating various down stream signalling pathways, inducing genes required for differentiation and for activation of osteoclasts. Exposure of monocytic precursors to M-CSF alone leads to differentiation into macrophages. Osteoclast differentiation was suppressed by granulocyte macrophage colony-stimulating factor (GM-CSF), resulting in mononuclear cells, lacking tartrate-resistant acid phosphatase (TRAP) and a bone resorptive phenotype. Further analysis determined GM-CSF dosage and temporal effects on osteoclast formation, where higher doses and earlier treatments of GM-CSF result in greater suppression of osteoclast formation. To understand the TRAP negative mononuclear cell phenotype, various osteoclast related markers and nuclear factors were tested using quantitative real-time PCR. GM-CSF suppressed the mRNA expression of osteoclast markers, including TRAP and cathepsin K (CTSK). CTSK is a cysteine protease, involved in osteoclast activity of bone resorption. Furthermore, GM-CSF down regulated the expression of critical osteoclast-related nuclear factors, including nuclear factor of activated T-cells, cytoplasmic (NFATcI), which has been identified as playing a critical role in osteoclast differentiation and ftinction in mice and to some extent in humans. The suppression of crucial osteoclast markers and transcription factors by GM-CSF indicated an overriding of the RANKL signal and possible switching of the cellular phenotype away from osteoclasts. To determine the cellular phenotype of GM-CSF driven cell differentiation, flow cytometry analysis was employed. As the cells visualised as dendritic cell like, CDIa, a dendritic cell surface marker, was selected for investigation. CDIa was highly expressed in GM-CSF, M-CSF and RANKL (GMR) treated cells and was absent in osteoclasts (M-CSF and RANKL treatment). The CDI a observations were indicative of GM-CSF overcoming the RANKL signal for osteoclastogenesis and directing differentiation to dendritic-like cells. To ftirther understand the osteoclastogenesis suppressive effect of GM-CSF, a 19,000 gene cDNA microarray assay was examined. The microarray experiment showed that the CC chemokine, monocyte chemotactic protein I (MCP-l), was profoundly repressed by GM-CSF. CC chemokines are chemoattractants that are induced during inflammation and recruit monocytes to the site of inflammation. MCP-l and other CC chemokines, RANTES (regulated on activation normal T cell expressed and secreted) and macrophage inflammatory protein I alpha (MIP I a) permitted formation of TRAP positive multinuclear cells in the absence of RANKL. However, these cells were negative for bone resorption. In the presence of RANKL, MCP-1 significantly increased the number of TRAP positive multinuclear bone resorbing osteoclasts (p= 5.7x 105, while RANTES and MIPI a mildly increased the number of bone resorbing TRAP positive multinuclear cells. Furthermore, CC chemokines, MCP-1, RANTES and MIP I a are all induced when authentic bone resorbing human osteoclasts differentiate from monocyte precursors in vitro following M-CSF-RANKL treatment. The addition of MCP- 1, RANTES or MIP I a appeared to reverse GM-CSF suppression of osteoclast formation, resulting in TRAP positive multinuclear cells. However, only MCP- I recovered the bone resorption phenotype, while other chemokines, RANTES or MTPIa did not. The cognate receptors for MCP-1, in particular, CCR2b and CCR4, were potently induced by RANKL (12.6 and 49-fold, p= 4.0x107 and 4.0x108, respectively), whereas the chemokine receptors for RANTES and MTP I a (CCR I and CCR5) were not regulated by RANKL. Chemokine treatment in the absence of RANKL also induced MCP- 1, RANTES and MIP I a. Unexpectedly, treatment with MCP-I in the absence of RANKL resulted in 458-fold induction of CCR4 (p I.0xI010), while RANTES treatment resulted in two fold repression (p= I .Ox ioj. Since CCR2b and CCR4 are cognate MCP-I receptors, these data support the existence of an MCP-I autocrine loop in human osteoclasts differentiated using RANKL. All three chemokines in the absence of RANKL can induce TRAP positive multinuclear cells that are negative for bone resorption. However, as MCP-I can significantly increase the number of osteoclast formation and recover the bone resorbing osteoclast phenotype from GM-CSF suppression, MCP-1 is the most potent chemokine involved in osteoclast formation. MCP-1 induced TRAP positive multinuclear cells were characterised and found to be positive for calcitonin receptor (CTR) and a number of other osteoclast markers, including NFATcI. As NFATcI is associated with osteoclast maturity in mice and has even been referred to as a master regulator of osteoclast differentiation and ftinction, a strong induction of NFATcI should theoretically allow bone resorption of MCP-l mediated TRAP positive multinuclear cells. Although great NFATcI mRNA induction and activated nuclear NFAT were observed, MCP-1 did not result in the formation of bone resorbing osteoclasts in the absence of RANKL. Despite the similar visual phenotype and expression of mature osteoclast markers TRAP and CTR when compared to osteoclasts, RANKL treatment was required for the MCP- I induced TRAP positive, CTR positive, multinuclear cells to possess bone resorption activity. This suggested that MCP-1 mediated TRAP positive multinuclear cells were primed for RANKL signal, to ftirther differentiate into authentic osteoclasts. The lack of bone resorption was ftirther correlated with a deficiency in expression of certain genes related to bone resorption, such as CTSK and matrix metalloproteinase 9 (MMP9) and integrin aV. Another observation with implications for absence of the bone resorptive activity in MCP- I cell was the absence or disruption of the F-actin ring structure, correlating with the lack of integrin aV mRNA expression. It was hypothesised that as MCP-1 mediated TRAP positive multinuclear cells possessed a high induction of CTR, the addition of calcitonin would block multinucleation. Indeed, the exogenous calcitonin blocked the MCP-I induced formation of TRAP positive, CTR positive, multinuclear cells as well as bone resorption activity in the osteoclast controls, indicating that calcitonin acts at two stages of osteoclast differentiation in the human PBMC model. These data suggest that RANKL-induced chemokines are involved in osteoclast differentiation at the stage of multinucleation of osteoclast precursors and provides a rationale for increased osteoclast activity in inflammatory conditions where chemokines are abundant. Furthermore, MCP-I induced TRAP positive, CTR positive multinuclear cells appear to represent an arrested stage in osteoclast differentiation, afler NFATcI induction and cellular ftision, but prior to the development of bone resorption activity and therefore, could be termed 'preosteoclasts'. Thesis (PhD Doctorate) Doctor of Philosophy (PhD) School of Medical Science Full Text

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2

Simcock, Wendy. "Parallel Analysis of Gene Expression: Bone Cells as a Model System." Thesis, Griffith University, 2005. http://hdl.handle.net/10072/365317.

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The use of comparative gene expression techniques has expanded considerably in recent years, especially with advances in microarray technology. In this project, a number of these techniques have been used to identify genes worthy of further research as potential mediators of bone cell differentiation and function. Bone is a tissue with many potential as yet unidentified regulatory molecules. The skeleton is constantly undergoing replacement, with old bone being degraded by osteoclasts, bone resorbing cells derived from the haematopoietic lineage, and replaced with new bone by osteoblasts, bone synthesizing cells derived from the mesenchymal stem cell lineage. When the rate of bone resorption exceeds the rate of bone synthesis, osteoporosis can occur. Osteoporosis is the most common form of disease affecting the skeleton, and one of the most common age-related diseases, and is a major social and economic burden. Recent studies have shown that cells of mesenchymal lineage are capable of adopting alternate differentiation fates, suggesting that cell-based therapies may be a useful therapeutic approach for this disease. Therefore, identification of molecular mechanisms involved in regulating the behavior and development of bone forming and bone resorbing cells is essential. The aim of this project, therefore, was to identify genes involved in various stages of bone cell differentiation using comparative gene expression techniques. The specific objectives of this project became: 1) to identify molecules expressed by osteoblasts which may increase or decrease bone synthesis; these may have potential for exploitation to treat bone loss in osteoporosis, or excess bone deposition in osteopetrosis; and 2) to identify molecules expressed by osteoclasts which may increase or decrease bone resorption; these may have potential for exploitation to treat excess bone deposition in osteopetrosis, or bone loss in osteoporosis. The first objective, identification of molecules expressed by osteoblasts involved in bone deposition, was addressed using three techniques: subtractive hybridization, DNA microarray analysis, and DNA macroarray analysis. These techniques were used to identify genes transcribed at different levels between foetal osteoblasts and fibroblasts. The key difference between DNA arrays, and subtractive hybridization, as techniques is that DNA arrays utilize a cDNA population fixed to a rigid medium as starting material. This means, therefore, that in order to identify a gene as being expressed, the gene must be present on the array, as a member of the cDNA library the original starting array was made from. This inhibits identification of truly novel transcripts, a bias which is removed in techniques such as subtractive hybridization. The technique of subtractive hybridization is used to identify genes transcribed at higher levels in one DNA sample compared with another. The subtractive hybridization technique described here was modified to enrich a foetal osteoblast phagemid library by removing phagemid which contain transcripts common to foetal osteoblasts and dermal fibroblasts, thus resulting in identification of genes expressed uniquely or at higher levels in osteoblasts. The technique identified 65 genes that were expressed either highly or specifically in osteoblasts when compared with fibroblasts. Some of the genes identified were found in multiple library clones, such as collagen and fibronectin, both of which are key structural components of bone, abundantly expressed by osteoblasts. Expression of some other identified genes had not previously been detected in osteoblasts, making them interesting targets for further investigation. Interesting genes revealed using this technique included prohibitin, leptin-receptor gene related protein, ornithine decarboxylase antizyme, amyloid precursor peptide and connective tissue growth factor. The usefulness of the technique was verified by performing real-time PCR to confirm the expression of these genes either specifically or abundantly in osteoblast cells. DNA array analysis was undertaken to identify transcripts previously identified in other tissues, but not investigated in bone cells to date. Micro and macroarray analysis was used to identify known genes that were over or underexpressed. The microarray comparison of fibroblasts and osteoblasts using cDNA differentially labeled with fluorescent dyes hybridized to glass microarrays, showed that the two cell types were very similar, with just 64 genes found to be regulated 5-fold or more- 37 were down-regulated in osteoblasts, and 27 were upregulated in osteoblasts, out of the 19,000 genes represented on the array. Genes shown to be significantly upregulated in osteoblasts by the 19k microarray included several neural proteins and transcription factors, while genes downregulated in osteoblasts included cell signal transducers and other transcriptional activity modifiers. The set of Atlas cDNA arrays consists of three pairs of arrays, with each pair containing 1176 genes. Two pairs of filters, or 2352 genes, were probed in the osteoblast/fibroblast comparison, while all three pairs, or 3528 genes, were probed in the osteoclast/macrophage comparison. The data from the DNA macroarray experiment, in which radioactively-labelled cDNA from osteoblasts and fibroblasts was hybridized to two separate sets of nylon arrays (ATLAS human cDNA arrays) showed that 12 out of the 2352 genes assayed were significantly regulated, with eight upregulated in osteoblasts, and four downregulated in osteoblasts. Genes upregulated in osteoblasts included transcription factor Dp-2, cAMP response element binding protein 1, and transcription factor ATF2. Genes down-regulated in osteoblasts included teratocarinoma derived growth factor, thymosin beta-10, and transcription factor 3. To address the second objective, and identify molecules expressed by osteoclasts involved in bone resorption, the DNA macroarray analysis approach was repeated. cDNA isolated from osteoclasts was compared with cDNA isolated from macrophages to identify genes differentially transcribed between these two cell types which differentiate from the same developmental lineage in vitro. DNA macroarray analysis, performed using radioactively labeled cDNA from osteoclasts and macrophages hybridized to ATLAS human cDNA arrays identified 53 genes as upregulated in osteoclasts, including GM-CSF Receptor, signalling molecule calmodulin 1, and transcription regulatory molecule Nuclear Factor of Activated T-cells (NFAT). Seventeen genes were shown to be downregulated in osteoclasts, including transcription factor zpf36, Integrin 5, and X-ray repair complementing protein. The studies described here suggested that osteoclasts and macrophages have more differentially expressed genes than osteoblasts and fibroblasts, with 1.98% of genes arrayed showing differential expression between osteoclasts and macrophages, but only 0.3% of genes arrayed showing differential expression between fibroblasts and osteoblasts. This suggests that the morphological differences between cell types may be a direct reflection of the molecular differences between them as well, as osteoblasts and fibroblasts are quite similar, while osteoclasts and macrophages are morphologically quite distinct. From this project, it would appear that comparisons of different populations of cells require the use of different techniques to yield the best results, and that the techniques of array analysis and subtractive hybridization may be best utilized in combination, rather than exclusively of each other. Many of the genes identified as differentially expressed between fibroblasts and osteoblasts using DNA arrays were fairly well-characterised 'house-keeping' type genes- metabolic, structural, and not specific or likely to play a significant role in the differentiation of osteoblasts, or the development of bone disease. One possible reason for this is that the arrays are too limited by the number of genes featured, to be able to detect many differences between similar cell types, where there are fewer differences to detect. In contrast, using the same macroarrays to compare the more distinct osteoclasts and macrophages resulted in identification of several interesting candidate genes, showing that some cell type comparisons can be performed adequately using this technology. By using the subtractive hybridization method to enrich a pre-existing phagemid library, any bias related to the genes able to be detected was removed. Although this technique requires manufacture of a cDNA library of the cell type of interest, it may be worthwhile for the comparison of similar cell types where sensitivity is an issue. In summary, this project used the techniques of subtractive hybridization and DNA macroarray and microarray analysis to detect genes showing differential expression between osteoblasts and fibroblasts, and used DNA macroarray analysis to detect genes differentially expressed between osteoclasts and macrophages. Of particular note were results for two interesting genes, amyloid precursor protein and ornithine decarboxylase antizyme. Amyloid Precursor Protein was identified as expressed in high levels in osteoblasts by subtractive hybridization, and real-time PCR studies later confirmed that it is expressed specifically in osteoblasts, and not at all in fibroblasts. Differential expression of ornithine decarboxylase antizyme was identified between both osteoclasts and macrophages, and fibroblasts and osteoblasts. Expression of antizyme results in destruction of ornithine decarboxylase, which is required for the production of polyamines. Degrading cells release spermidine, a polyamine, which attracts macrophages. It is possible that differential regulation of the inhibitory antizyme may be an important distinction between the function of macrophages as general tissue and debris scavenging cells, and osteoclasts, which specifically degrade bone. This study has identified some genes which further studies may show to be important regulators of cellular differentiation and behaviour. Thesis (PhD Doctorate) Doctor of Philosophy (PhD) School of Health Sciences Full Text

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Granholm, Susanne. "The calcitonin gene family of peptides : receptor expression and effects on bone cells." Doctoral thesis, Umeå : Univ, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1571.

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Stiehler, Maik, Juliane Rauh, Cody Bünger, et al. "Large-scale gene expression profiling data of bone marrow stromal cells from osteoarthritic donors." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2017. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-217567.

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This data article contains data related to the research article entitled, "in vitro characterization of bone marrow stromal cells from osteoarthritic donors" [1]. Osteoarthritis (OA) represents the main indication for total joint arthroplasty and is one of the most frequent degenerative joint disorders. However, the exact etiology of OA remains unknown. Bone marrow stromal cells (BMSCs) can be easily isolated from bone marrow aspirates and provide an excellent source of progenitor cells. The data shows the identification of pivotal genes and pathways involved in osteoarthritis by comparing gene expression patterns of BMSCs from osteoarthritic versus healthy donors using an array-based approach.

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Peake, Matthew. "Regulatory pathways involved in mechanical induction of c-fos gene expression in bone cells." Thesis, Keele University, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.392159.

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The regulatory pathways involved in the rapid response of the AP-I transcription factor component, c-fos, to mechanical load in human primary osteoblast-like (HOB) cells and the human MO-63 bone cell line were investigated using a four-point bending model. HOB and MO-63 cells showed up regulation of c-fos expression after I-hour loading on fibronectin and collagen type I substrates; however, MO-63 cells did not respond on laminin YIGSR substrates. It was suggested that RGD mediated integrin interactions might be non-essential for the mechanical induction of c-fos at certain time points as indicated by a lack of inhibition associated with ROD-peptide treatment (however, evidence of the inhibitory nature of soluble RGD-peptides at these time points may require further examination). β1 integrin mediated interactions are critical as induction was completely blocked by anti-β1 integrin antibodies. The role of calcium signalling pathways was demonstrated by blocking up regulation with addition of EGTA, which chelates extracellular Ca2+, and gadolinium, which inhibits stretch-activated channels. L-type calcium channels may also be contributory but not essential, as addition of the L-type channel blocker, nifedipine inhibited the response, but not completely. Further evidence for involvement of calcium pathways followed addition of the Ca2+ ionophore A-23187 that induced temporally identical up regulation without loading. Protein kinase (C) mediated pathways were also shown to be critical, as shown by abolition of the response following H7 -dichloride treatment. Prostaglandin signalling pathways were non-essential, but were implicated in maximising load induction of c-fos as indicated by indomethacin induced inhibition. C-fos promoter analysis indicated that the major CRE is not essential for mechanically induced transcriptional activation of c-fos. An SRE containing promoter fragment appears to play a key role in this induction but is also not essential, indicating that multiple response elements are required. These results therefore demonstrate the essential nature of calcium, integrin and protein kinase mediated pathways in the induction of the early transcriptional mechanoresponse of bone, which are mediated by multiple response elements in the c-fos promoter.

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Stiehler, Maik, Juliane Rauh, Cody Bünger, et al. "Large-scale gene expression profiling data of bone marrow stromal cells from osteoarthritic donors." Elsevier, 2016. https://tud.qucosa.de/id/qucosa%3A30119.

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This data article contains data related to the research article entitled, 'in vitro characterization of bone marrow stromal cells from osteoarthritic donors' [1]. Osteoarthritis (OA) represents the main indication for total joint arthroplasty and is one of the most frequent degenerative joint disorders. However, the exact etiology of OA remains unknown. Bone marrow stromal cells (BMSCs) can be easily isolated from bone marrow aspirates and provide an excellent source of progenitor cells. The data shows the identification of pivotal genes and pathways involved in osteoarthritis by comparing gene expression patterns of BMSCs from osteoarthritic versus healthy donors using an array-based approach.

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Montecino, Martin A. "Chromatin Structure of the Rat Osteocalcin Gene Promoter in Bone-Derived Cells." eScholarship@UMMS, 1995. https://escholarship.umassmed.edu/gsbs_diss/33.

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Transcription of the osteocalcin gene, which encodes a bone-specific 10 kDa protein, is controlled by the coordinated utilization of modularly organized basal and hormone-responsive enhancer elements. Activation of these sequences involves the interaction of specific transcription factors to these promoter elements. It is becoming increasingly accepted that nuclear architecture provides a basis for support of tightly regulated modulation of cell growth and tissue-specific transcription which is required for the onset and progression of differentiation. Thus packaging of DNA as chromatin can facilitate the cooperative interaction between activities of independent regulatory elements that contribute to the level of transcription. Here, we show that a specific nucleosomal organization supports the constitutive expression of the osteocalcin gene in ROS 17/2.8 rat osteosarcoma cells and that chromatin remodeling directly correlates with the developmentally regulated transcriptional activation of this gene in normal diploid osteoblasts. By combining DNase I, micrococcal nuclease, and specific restriction endonuclease digestion analysis, we observed that the presence of DNase I hypersensitive sites (proximal: -170 to -70, and distal: -600 to -400) and a selective nucleosome positioning over the osteocalcin gene promoter are directly associated with developmentally stage-specific transcriptional activation in bone-derived cells. In addition, we found that chromatin hyperacetylation prevents a key transition in the chromatin structure which is required for the formation of the distal DNase I hypersensitive site. This transition involves the interaction of specific nuclear factors and is necessary for the subsequent ligand-dependent binding of the vitamin D receptor complex. Finally, we have established a requirement for sequences residing in the proximal region of the osteocalcin gene promoter for both formation of the proximal hypersensitive site and basal transcriptional activity. Our approach was to assay nuclease accessibility in ROS 17/2.8 cell lines stably transfected with promoter deletion constructs driving expression of a CAT reporter gene.

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Byers, Benjamin Allen. "In vitro and in vivo characterization of a cell source for bone tissue engineering applications primary bone marrow stromal cells overexpressing the osteoblast-specific transcriptional activator Runx2/Cbfa1 /." Available online, Georgia Institute of Technology, 2003:, 2003. http://etd.gatech.edu/theses/available/etd-02102004-164825/unrestricted/byers%5Fbenjamin%5Fa%5F200405%5Fphd.pdf.

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Thesis (Ph. D.)--Mechanical Engineering, Georgia Institute of Technology, 2004. Joseph M. LeDoux, Committee Member ; Julia E. Babensee, Committee Member ; Robert E. Guldberg, Committee Member ; Andres J. Garcia, Committee Chair ; Grace K. Pavlath, Committee Member ; Barbara D. Boyan, Committee Member. Includes bibliographical references.

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Krüger-Almerén, Anders. "RP59, a novel stem cell protein and mapping of its expression /." Stockholm, 2002. http://diss.kib.ki.se/2002/91-7349-246-9.

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Lee, Kate L. "Protein and gene expression analyses in bone marrow stem cells mediated restoration of myocardium after ischemic insult." Thesis, Queen Mary, University of London, 2010. http://qmro.qmul.ac.uk/xmlui/handle/123456789/559.

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Myocardial Infarction (MI) is caused by occlusion of the coronary artery following atheromatous plaque rupture, the subsequent ischemia in the myocardium leads to myocyte necrosis unless treated quickly. Bone marrow derived stem cell treatment is a promising therapy for improving the outcome of patients with MI. The aim of this thesis was to study myocardial protein and gene expression changes in a rat ischemia/reperfusion (I/R) model in order to look for potential repair mechanisms of the myocardium triggered by endogenous bone marrow mononuclear cells (BMMNCs). Rat myocardial samples were obtained from three experimental groups: one group had a sham operation, the other two groups had undergone myocardial I/R injury induced by left anterior descending (LAD) coronary artery ligation followed by treatment with either a BMMNC preparation or PBS. Comparative proteomic analyses were carried out using 2D electrophoresis; differentially expressed proteins were identified using LC-MS/MS. Western blotting was used to confirm the most significant findings including expression of 14-3-3 epsilon protein. Global comparative gene expression profiling was performed using Illumina RatRef12 BeadChips and QPCR was used to validate the top results. Bioinformatic tools were used to assess the biology of the differentially expressed genes and proteins. Thirty-seven proteins were found to be differentially expressed in I/R injury compared to sham. These were primarily sarcomeric, energy production or stress response proteins and most were down-regulated. Expression levels were ‘corrected’ by BMMNC treatment for many of these proteins. Over 1500 genes were affected by I/R injury, 20 were affected by BMMNC treatment, and many of these were related to inflammation and apoptosis signalling and responses. The 14-3-3 epsilon protein was chosen for follow-up work as it presented as a good candidate for mechanistic involvement. This protein has many roles including interactions with the proapoptotic BCL2-associated agonist of cell death (Bad) protein. Western blotting was used to look at Bad expression and found it to be significantly increased in the Page 3 treatment group, although I could not reliably measure the expression of phosphorylated (serine 136) form of Bad. A preliminary pull-down assay was performed to look for binding partners of 14-3-3 epsilon. Two ATP synthase subunits, one of which is known to bind 14-3-3 epsilon, a protein involved in fatty acid β-oxidation and a protein of unknown function were found to bind. Further work will be required to follow up these findings and ascertain the exact role of 14-3- 3 epsilon in cardioprotection. In summary, my data supports the power of profiling methods to derive new candidates for a role in repair mechanisms in this therapeutic model.

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Chen, Chih-Yeh [Verfasser], and Philipp [Akademischer Betreuer] Beckhove. "Induction of autoreactive regulatory T cells through promiscuous gene expression by bone marrow-resident plasma cells / Chih-Yeh Chen ; Betreuer: Philipp Beckhove." Heidelberg : Universitätsbibliothek Heidelberg, 2019. http://d-nb.info/119530255X/34.

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Xiao, Qi. "Bone morphogenetic proteins (BMPS) mediate cellular response and regulate neural stem cell differentiation after acute spinal cord injury in the adult mice." Click to view the E-thesis via HKUTO, 2008. http://sunzi.lib.hku.hk/hkuto/record/B41290446.

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Sugino, Noriko. "Early osteoinductive human bone marrow mesenchymal stromal/stem cells support an enhanced hematopoietic cell expansion with altered chemotaxis- and adhesion-related gene expression profiles." Kyoto University, 2016. http://hdl.handle.net/2433/215424.

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Final publication is available at http://www.sciencedirect.com/science/article/pii/S0006291X15310664 Kyoto University (京都大学) 0048 新制・課程博士 博士(医学) 甲第19598号 医博第4105号 新制||医||1014(附属図書館) 32634 京都大学大学院医学研究科医学専攻 (主査)教授三森経世, 教授開祐司, 教授妻木範行 学位規則第4条第1項該当

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Burk, Janina, Claudia Gittel, Sandra Heller, et al. "Gene expression of tendon markers in mesenchymal stromal cells derived from different sources." Universitätsbibliothek Leipzig, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-157823.

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Background: Multipotent mesenchymal stromal cells (MSC) can be recovered from a variety of tissues in the body. Yet, their functional properties were shown to vary depending on tissue origin. While MSC have emerged as a favoured cell type for tendon regenerative therapies, very little is known about the influence of the MSC source on their properties relevant to tendon regeneration. The aim of this study was to assess and compare the expression of tendon extracellular matrix proteins and tendon differentiation markers in MSC derived from different sources as well as in native tendon tissue. MSC isolated from equine bone marrow, adipose tissue, umbilical cord tissue, umbilical cord blood and tendon tissue were characterized and then subjected to mRNA analysis by real-time polymerase chain reaction. Results: MSC derived from adipose tissue displayed the highest expression of collagen 1A2, collagen 3A1 and decorin compared to MSC from all other sources and native tendon tissue (p < 0.01). Tenascin-C and scleraxis expressions were highest in MSC derived from cord blood compared to MSC derived from other sources, though both tenascin-C and scleraxis were expressed at significantly lower levels in all MSC compared to native tendon tissue (p < 0.01). Conclusions: These findings demonstrate that the MSC source impacts the cell properties relevant to tendon regeneration. Adipose derived MSC might be superior regarding their potential to positively influence tendon matrix reorganization.

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Banerjee, Chaitali. "The Osteocalcin Gene: Transcriptional Elements and Factors Regulating TGF-β1 Responsiveness and Tissue-Specific Expression in Bone Cells: A Dissertation". eScholarship@UMMS, 1998. http://escholarship.umassmed.edu/gsbs_diss/145.

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Osteocalcin (OC) is a bone specific protein expressed during differentiation and mineralization of the bone extracellular matrix. The Osteocalcin gene is transcriptionally regulated by several basal, hormone- and cytokine-responsive elements. To address the potential role of TGF-β1 regulation and tissue-specific expression of the OC gene, we defined regulatory elements and factors mediating the transcriptional activity of the rat OC (rOC) promoter. TGF-β1 modulates the differentiation of cells of the osteogenic lineage and downregulates the osteoblast-specific expression of OC. By promoter deletion and mutational analyses, a TGF-β1 responsive element at nt -146 to -139 and a contiguous tissue-specific enhancer element at nt -136 to -130 on the rOC promoter were identified. These studies show that Fra-2, a member of the AP-1 family of proteins, binds to the TGF-β1 responsive element and activates basal OC expression. TGF-β1 induced phosphorylation of Fra-2 inhibits this activation, resulting in repression of OC gene transcription. The tissue-specific enhancer element contiguous to the TGF-β1 responsive element contains an AML (Cbfa) binding sequence. This element, designated OC Box II, contributes to 75% of the basal OC promoter activity and forms an osteoblast-specific protein-DNA complex in in-vitro assays. The activation potential of this binding sequence was established by overexpressing AML (Cbfa) transcription activator proteins in osteoblastic as well as in non-osseous cell lines. Interestingly, overexpression not only enhances rOC promoter activity in osteoblasts but also mediates promoter activity in a non-osseous human fibroblastic cell-line. Subsequently, we identified AML-3 (Cbfa1) as the major AML family member present in the osteoblast specific complex and demonstrate that AML-3 (Cbfa1) is expressed predominantly as a 5.4 kb transcript in rat bone tissues. Finally, to establish the functional involvement of AML (Cbfa) transcription factors in osteoblast differentiation, we utilized antisense strategies to demonstrate that blocking expression of all AML (Cbfa) related genes in primary osteoblast cultures significantly decreased several parameters which are linked to differentiation of normal diploid osteoblasts. These results indicate that AML-3 (Cbfa1) is a key transcription factor in bone cells and that the activity of the AML (Cbfa) family of proteins is required for completion of osteoblast differentiation.

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Santiago-Torres, Juan E. "Fetal Mesenchymal Stem Cells Achieve Greater Gene Expression in Vitro, but Less Effective Osteoinduction in Vivo than Adult Mesenchymal Stem Cells." The Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1404561922.

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Xiao, Qi, and 肖琦. "Bone morphogenetic proteins (BMPS) mediate cellular response and regulate neural stem cell differentiation after acute spinal cordinjury in the adult mice." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2008. http://hub.hku.hk/bib/B41290446.

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Barnes, Calvin Langston Toure. "C-mpl Expression in Osteoclast Progenitors: A Novel Role for Thrombopoietin in Regulating Osteoclast Development." Yale University, 2006. http://ymtdl.med.yale.edu/theses/available/etd-06262006-123750/.

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A new paradigm has evolved in which multiple regulatory interactions between the skeletal and hematopoietic systems have been identified. Previous studies have demonstrated that megakaryocytes (MK) play a dual role in skeletal homeostasis by stimulating osteoblast proliferation and simultaneously inhibiting osteoclast (OC) development. Here we identify a novel regulatory pathway in which the main MK growth factor, thrombopoietin (TPO), directly regulates osteoclastogenesis. To study the role of TPO in OC development, spleen or bone marrow (BM) cells (2x10[exponent]6 cells/ml) or BM macrophages (BMM, 1x10[exponent]5 cells/ml) from C57BL/6 mice , as a source of OC precursors, were cultured with M-CSF (30 ng/ml) and RANKL (50 ng/ml) to induce OC formation. TPO (0.1-1000 ng/ml) and/or primary MK (0-0.5%), derived from C57BL/6 fetal livers, were titrated into these cultures and OC were identified as tartrate resistant acid phosphatase positive (TRAP+) giant cells with >3 nuclei. There was a significant, up to 15-fold reduction in OC formed when MK were added to all OC generating cultures, p < 0.001. Moreover, if OC generating cultures did not contain MK or MK progenitors, TPO treatment significantly enhanced OC formation up to six-fold, p < 0.01. This data demonstrates that MK are responsible for the inhibition of OC formation and that in cultures containing MK or MK progenitors such as BM or spleen cells, that TPO acts indirectly to inhibit OC formation by stimulating megakaryopoiesis, whereas in the absence of MK or MK progenitors TPO directly enhances OC formation. This conclusion is further supported by Real-Time PCR data which demonstrates that OC progenitors express c-mpl, the TPO receptor, albeit at low levels when compared to expression of c-mpl on MK. Finally, we have begun to dissect the c-mpl signaling pathway in OC progenitors. We have found that TPO induces tyrosine phosphorylation of several specific cellular proteins in the JAK/STAT pathway. Thus, TPO acts in a somewhat paradoxical manner by inhibiting OC formation through the stimulation of MK, while simultaneously playing a direct role in enhancing osteoclastogenesis.

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Mareddy, Shobha R. "Characterization of bone marrow stromal clonal populations derived from osteoarthritis patients." Thesis, Queensland University of Technology, 2008. https://eprints.qut.edu.au/17151/1/Shobha_Reddy_Mareddy_Thesis.pdf.

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This work is concerned with the characterization of mesenchymal stem cells (MSC) specifically from bone marrow samples derived from patients with osteoarthritis (OA). The multilineage potential of mesenchymal stem cells as well as their ease of exvivo expansion makes these cells an attractive therapeutic tool for applications such as autologous transplantation and tissue engineering. Bone marrow is considered a source of MSC. However, there is a general assumption that the occurrence of MSCs and their activity in bone marrow diminishes with age and disease. This prompted us to isolate and identify multipotential and self-renewing cells from patients with the degenerative disease osteoarthritis, with the view of using these cells for autologous cell therapies. It is therefore of great potential benefit to investigate the isolation and characterization of stem cell/progenitors from bone marrow samples of patients with osteoarthritis in greater detail. We employed a single cell clone culture method in order to develop clonal cell populations from three bone marrow samples and characterized them based on their proliferation and differentiation capabilities. The clonal populations were grouped into fast-growing and slow-growing clones based on their proliferation rates. The fastgrowing clones displayed 20-30% greater proliferation rate than the slow-growing clones. The study also revealed that the proliferation rates were directly proportional to their differentiation capacities. Most of the fast-growing clones were found to be tripotential for osteogenic, chondrogenic and adipogenic lineages, whereas the slow growing clones were either uni or bipotential. Flow cytometry analysis for the phenotype determination using putative MSC surface markers did not reveal any difference between the two clonal populations indicating a need for further molecular studies. Two approaches were employed to further investigate the molecular processes involved in the existence of such varying populations. In the first method gene expression studies were performed between the fast-growing (n=3) and slow-growing (n=3) clonal populations to identify potential genetic markers associated with cell 'sternness' using the Stem Cell RT2 ProfilerTM PCR Array comprising a series of 84 genes related to stem cell pathways. Ten genes were identified to be commonly and significantly over represented in the fast-growing stem cell clones when compared to slow-growing clones. This included expression of transcripts beyond MSC lineage specification such as SOX2, NOTCH1 and FOXA2 which signified that stem cell maintenance requires a coordinated regulation by multiple signalling pathways. The second study involved an extensive protein expression profiling of the fast growing (n=2) and slow growing (n=2) clonal populations using off-line Two Dimensional Liquid Chromatography (2D-LC)/Matrix-Assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry (MS). A total of 67 proteins were identified, of which 11 were expressed at significantly different levels between the subpopulations. Protein ontology revealed these proteins to be associated with cellular organization, cytokinesis, signal transduction, energy pathways and cell stress response. Of particular interest was the differential presentation of the proteins calmodulin, tropomyosin and caldesmon between fast- and slow-growing clones. Based on their reported roles in the regulation of cell proliferation and maintenance of cell integrity, we draw an association between their expression and the altered status in which the subpopulations exist. Based on our observations, these proteins may be prospective molecular markers to distinguish between the fast-growing and slow-growing subpopulations. In summary, this study demonstrated the existence of potential stem cells of therapeutic importance in spite of a supposedly smaller stem cell compartment in patients with osteoarthritis. Furthermore, the differentially expressed genes between the sub-populations highlight the 'sternness' of the potential clones, an observation supported by the expression of proteins which act as effective modulators in the maintenance of cell integrity and cell cycle regulation. This study provides a basis for more detailed investigations in search of selective cell surface markers

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20

Mareddy, Shobha R. "Characterization of bone marrow stromal clonal populations derived from osteoarthritis patients." Queensland University of Technology, 2008. http://eprints.qut.edu.au/17151/.

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This work is concerned with the characterization of mesenchymal stem cells (MSC) specifically from bone marrow samples derived from patients with osteoarthritis (OA). The multilineage potential of mesenchymal stem cells as well as their ease of exvivo expansion makes these cells an attractive therapeutic tool for applications such as autologous transplantation and tissue engineering. Bone marrow is considered a source of MSC. However, there is a general assumption that the occurrence of MSCs and their activity in bone marrow diminishes with age and disease. This prompted us to isolate and identify multipotential and self-renewing cells from patients with the degenerative disease osteoarthritis, with the view of using these cells for autologous cell therapies. It is therefore of great potential benefit to investigate the isolation and characterization of stem cell/progenitors from bone marrow samples of patients with osteoarthritis in greater detail. We employed a single cell clone culture method in order to develop clonal cell populations from three bone marrow samples and characterized them based on their proliferation and differentiation capabilities. The clonal populations were grouped into fast-growing and slow-growing clones based on their proliferation rates. The fastgrowing clones displayed 20-30% greater proliferation rate than the slow-growing clones. The study also revealed that the proliferation rates were directly proportional to their differentiation capacities. Most of the fast-growing clones were found to be tripotential for osteogenic, chondrogenic and adipogenic lineages, whereas the slow growing clones were either uni or bipotential. Flow cytometry analysis for the phenotype determination using putative MSC surface markers did not reveal any difference between the two clonal populations indicating a need for further molecular studies. Two approaches were employed to further investigate the molecular processes involved in the existence of such varying populations. In the first method gene expression studies were performed between the fast-growing (n=3) and slow-growing (n=3) clonal populations to identify potential genetic markers associated with cell 'sternness' using the Stem Cell RT2 ProfilerTM PCR Array comprising a series of 84 genes related to stem cell pathways. Ten genes were identified to be commonly and significantly over represented in the fast-growing stem cell clones when compared to slow-growing clones. This included expression of transcripts beyond MSC lineage specification such as SOX2, NOTCH1 and FOXA2 which signified that stem cell maintenance requires a coordinated regulation by multiple signalling pathways. The second study involved an extensive protein expression profiling of the fast growing (n=2) and slow growing (n=2) clonal populations using off-line Two Dimensional Liquid Chromatography (2D-LC)/Matrix-Assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry (MS). A total of 67 proteins were identified, of which 11 were expressed at significantly different levels between the subpopulations. Protein ontology revealed these proteins to be associated with cellular organization, cytokinesis, signal transduction, energy pathways and cell stress response. Of particular interest was the differential presentation of the proteins calmodulin, tropomyosin and caldesmon between fast- and slow-growing clones. Based on their reported roles in the regulation of cell proliferation and maintenance of cell integrity, we draw an association between their expression and the altered status in which the subpopulations exist. Based on our observations, these proteins may be prospective molecular markers to distinguish between the fast-growing and slow-growing subpopulations. In summary, this study demonstrated the existence of potential stem cells of therapeutic importance in spite of a supposedly smaller stem cell compartment in patients with osteoarthritis. Furthermore, the differentially expressed genes between the sub-populations highlight the 'sternness' of the potential clones, an observation supported by the expression of proteins which act as effective modulators in the maintenance of cell integrity and cell cycle regulation. This study provides a basis for more detailed investigations in search of selective cell surface markers

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Rony, R. M. Imtiaz Karim. "Transcriptional characterization of osteogenic and adipogenic differentiation of human bone marrow derived mesenchymal stem cells in 2D and 3D peptide hydrogel culture system." Wright State University / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=wright1536841298659517.

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Greig, Kylie Tara. "Critical roles for the transcription factor c-Myb in early B cell development /." Connect to thesis, 2009. http://repository.unimelb.edu.au/10187/3824.

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Thesis (Ph.D.)--University of Melbourne, The Walter and Eliza Hall Institute of Medical Research, The Division of Immunology and the Division of Molecular Medicine, Dept. of Medical Biology, Faculty of Medicine and Dentistry, 2009. Typescript. Includes bibliographical references (p. 133-165)

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23

Tsourdi, Elena, Juliane Salbach-Hirsch, Martina Rauner, et al. "Glycosaminoglycans and their sulfate derivatives differentially regulate the viability and gene expression of osteocyte-like cell lines." Sage, 2014. https://tud.qucosa.de/id/qucosa%3A35689.

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Collagen and glycosaminoglycans, such as hyaluronan and chondroitin sulfate, are the major components of bone extracellular matrix, and extracellular matrix composites are being evaluated for a wide range of clinical applications. The molecular and cellular effects of native and sulfatemodified glycosaminoglycans on osteocytes were investigated as critical regulators of bone remodeling. The effects of glycosaminoglycans on viability, necrosis, apoptosis, and regulation of gene expression were tested in two osteocyte-like cell lines, the murine MLO-Y4 and the rat UMR 106-01 cells. Glycosaminoglycans were non-toxic and incorporated by osteocytic cells. In MLO-Y4 cells, sulfation of glycosaminoglycans led to a significant inhibition of osteocyte apoptosis, 42% inhibition for highly sulfated chondroitin sulfate and 58% for highly sulfated hyaluronan, respectively. Cell proliferation was not affected. While treatment with highly sulfated chondroitin sulfate increased cell viability by 20% compared to the native chondroitin sulfate. In UMR 106- 01 cells, treatment with highly sulfated hyaluronan reduced the receptor activator of nuclear factor-κB ligand/osteoprotegerin ratio by 58% compared to the non-sulfated form, whereas highly sulfated chondroitin sulfate led to 60% reduction in the receptor activator of nuclear factor-κB ligand/osteoprotegerin ratio in comparison to the native chondroitin sulfate. The expression of SOST, the gene encoding sclerostin, was reduced by 50% and 45% by highly sulfated hyaluronan and chondroitin sulfate, respectively, compared to their native forms. The expression of BMP- 2, a marker of osteoblast differentiation, was doubled after treatment with the highly sulfated hyaluronan in comparison to its native form. In conclusion, highly sulfated glycosaminoglycans inhibit osteocyte apoptosis in vitro and promote an osteoblast-supporting gene expression profile.

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Melo, Willian Morais de. "Osso autógeno associado a osso bovino inorgânico (GenOx Inorg®) para aumento do soalho do seio maxilar e instalação de implantes: análise comparativa do potencial osteogênico de culturas de células derivadas do sítio doador e do sítio de implantação." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/58/58136/tde-10082012-163454/.

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Objetivos: O objetivo desse estudo foi avaliar comparativamente o potencial osteogênico in vitro de células obtidas do ramo mandibular (RM, área doadora) e do seio maxilar enxertado com uma mistura de RM e osso bovino inorgânico (OBI), previamente à instalação de implantes de titânio (SM, sítio do seio maxilar enxertado). Material e Métodos: As células foram obtidas de três pacientes submetidos a procedimentos de aumento do soalho do seio maxilar com a proporção de 1:1 de RM e OBI (GenOx Inorg®). No momento da realização dos enxertos no seio maxilar e após 08 meses, antes da inserção dos implantes de titânio, fragmentos ósseos foram colhidos do RM e do SM, respectivamente, e submetidos à digestão enzimática com tripsina e colagenase para obtenção de células primárias. As células foram subcultivadas e crescidas sob condições osteogênicas por até 21 dias, tendo sido avaliados os seguintes parâmetros: proliferação/viabilidade celular, expressão gênica de marcadores osteoblásticos, atividade de fosfatase alcalina (ALP) e conteúdo de cálcio, por extração do vermelho de Alizarina. Culturas primárias derivadas do RM foram expostas ao GenOx Inorg® por 7 dias, quando se avaliou a atividade de ALP. Os resultados foram comparados por ANOVA two-way, seguido do teste de Tukey, ou pelo teste de Mann-Whitney. Resultados: Culturas do SM exibiram uma redução significante do potencial osteogênico se comparado ao de culturas do RM, com um aumento progressivo na proliferação celular associado a uma redução da expressão dos marcadores osteoblásticos, da atividade de ALP e do conteúdo de cálcio. A exposição do GenOx Inorg® às células primárias derivadas do RM inibiram a atividade de ALP. Conclusão: Esses resultados sugerem que o uso do GenOx Inorg® em associação a fragmentos do RM para aumento do soalho do seio maxilar inibe a diferenciação de células osteoblásticas no sítio de inserção de implantes de titânio após 8 meses de enxertia. Objectives: This study aimed to comparatively evaluate the in vitro osteogenic potential of cells obtained from the mandibular ramus (MR, autogenous bone donor site) and from the maxillary sinus bone grafted with a mixture of anorganic bovine bone (ABB) and MR prior to titanium implant placement (MS, grafted implant site). Material and methods: Cells were obtained from three patients subjected to maxillary sinus floor augmentation with a 1:1 mixture of ABB (GenOx Inorg®) and MR. At the time of the sinus lift procedure and after 8 months, prior to implant placement, bone fragments were taken from MR and MS, respectively, and subjected to trypsin-collagenase digestion for primary cell culturing. Subcultured cells were grown under osteogenic condition for up 21 days and assayed for proliferation/viability, osteoblast marker mRNA levels, alkaline phosphatase (ALP) activity and calcium content/Alizarin red staining. ALP activity was also determined in primary explant cultures exposed to GenOx Inorg® (1:1 with MR) for 7 days. Data were compared using the two-way ANOVA followed by the Tukey test; otherwise, the Mann-Whitney test was used. Results: MS cultures exhibited a significantly lower osteogenic potential compared with MR cultures, with a progressive increase in cell proliferation together with a downregulation of osteoblast markers, reduced ALP activity and calcium content. Exposure of MR-derived primary cultures to GenOx Inorg® inhibited ALP activity. Conclusion: These results suggest that the use of GenOx Inorg® in combination with MR fragments for maxillary sinus floor augmentation inhibits the osteoblast cell differentiation at the implant site in the longterm.

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Oliveira, Lucila Habib Bourguignon [UNESP]. "Avaliação do perfil de expressão gênica de células CD34+ e células CD CD133+ isoladas de medula óssea e de sangue de cordão umbilical." Universidade Estadual Paulista (UNESP), 2008. http://hdl.handle.net/11449/86617.

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Made available in DSpace on 2014-06-11T19:22:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-08-19Bitstream added on 2014-06-13T19:07:44Z : No. of bitstreams: 1 oliveira_lhb_me_arafcf.pdf: 553720 bytes, checksum: b0f8f04b07802f8bf467e43259c87fba (MD5) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Universidade Estadual Paulista (UNESP) A maior expressão de alvos transcricionais e componentes da via NFkB é uma característica distintiva das células-tronco hematopoéticas (CTH) CD34+ de sangue de cordão umbilical (SCU) comparadas às CTH CD34+ de medula óssea (MO) e pode estar relacionada com o estado mais primitivo das CTH dos neonatos. No entanto, as células CD34+ são um grupo heterogêneo de célulastronco (CT) e progenitoras em diferentes estágios de maturação e diferenças na composição celular entre MO e SCU poderiam contribuir para os resultados mencionados. Estudos recentes têm identificado o marcador de superfície CD133, como um marcador de CT mais primitivas, expresso em uma subpopulação de células CD34bright, com um sugestivo potencial de hemangioblasto. Com o objetivo de caracterizar a composição celular de MO e SCU e identificar mecanismos moleculares envolvidos com a maior primitividade das células CD133+, propusemos avaliar o perfis imunofenotípico (quanto à expressão de CD34 e CD133) por citometria de fluxo e de expressão gênica de células CD34+ e células CD133+ selecionadas imunomagneticamente, de ambas as fontes, pelas técnicas de microarray e PCR em tempo real. Nossos resultados revelaram que enquanto a maioria das células CD133+ são CD34+, independente da fonte, as células CD34+ de SCU possuem uma porcentagem significativamente maior de células CD133+ do que às células CD34+ de MO. A análise de clusterização revelou que as células CD133+ de MO se agrupam com as células de SCU (CD34+ e CD133+), enquanto as CD34+ de MO aparecem como um grupo distinto. A comparação dos perfis de expressão gênica entre as células CD133+ e as células CD34+, revelou a hiper-expressão... A higher expression of transcription targets and components of the NF-kB pathway is a distinctive feature of umbilical cord blood (UCB) CD34+ hematopoietic stem cells (HSC) when compared to bone marrow (BM) CD34+ HSC and this could be related to the more primitive state of the newborn’s HSC. However, CD34+ cells represent a heterogeneous group of cells composed by stem and progenitors cells in different developmental stages, and differences in cellular composition between both sources could contribute for these finding. The surface marker CD133 has been identified as a very primitive marker, expressed in a subpopulation of CD34bright, with a proposal hemangioblast potential. Thus, in attempt to better characterize the cellular composition of UCB and BM and to identify molecular mechanisms related to the more primitive characteristics of CD133+ cells, we proposed to evaluate the immunophenotypic profile (expression of CD34 and CD133) by flow-cytometry and the gene expression profiles of immunomagnetically selected CD34+ and CD133+ cells, from both sources, by microarray and Real time PCR. Our results highlighted that, while almost all CD133+ cells are CD34+ independently of the evaluated source, the UCB CD34+ cells showed a significantly higher proportion of CD133 expression, compared to BM CD34+ cells. After obtaining the expression profiles from distinct HSC pooled samples generated by microarrays, cluster analysis showed that BM CD133+ cells preferentially grouped with UCB cells (CD34+ and CD133+) instead of BM CD34+ cells, which appeared as a very distinct profile The comparison between CD133+ and CD34+ samples revealed the over-expression of 47 transcriptional factors (TF) in CD133+ cells, many of them well-known and related... (Complete abstract click electronic access below)

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Bewry, Nadine N. "STAT3 Contributes to Resistance Towards BCR-ABL Inhibitors in a Bone Marrow Microenvironment Model of Drug Resistance in Chronic Myeloid Leukemia Cells." [Tampa, Fla] : University of South Florida, 2009. http://purl.fcla.edu/usf/dc/et/SFE0002892.

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Oliveira, Lucila Habib Bourguignon. "Avaliação do perfil de expressão gênica de células CD34+ e células CD CD133+ isoladas de medula óssea e de sangue de cordão umbilical /." Araraquara : [s.n.], 2008. http://hdl.handle.net/11449/86617.

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Orientador: Dimas Tadeu Covas Banca: Dimas Tadeu Covas Banca: Rodrigo Alexandre Panepucci Banca: Haroldo Wilson Moreira Resumo: A maior expressão de alvos transcricionais e componentes da via NFkB é uma característica distintiva das células-tronco hematopoéticas (CTH) CD34+ de sangue de cordão umbilical (SCU) comparadas às CTH CD34+ de medula óssea (MO) e pode estar relacionada com o estado mais primitivo das CTH dos neonatos. No entanto, as células CD34+ são um grupo heterogêneo de célulastronco (CT) e progenitoras em diferentes estágios de maturação e diferenças na composição celular entre MO e SCU poderiam contribuir para os resultados mencionados. Estudos recentes têm identificado o marcador de superfície CD133, como um marcador de CT mais primitivas, expresso em uma subpopulação de células CD34bright, com um sugestivo potencial de hemangioblasto. Com o objetivo de caracterizar a composição celular de MO e SCU e identificar mecanismos moleculares envolvidos com a maior primitividade das células CD133+, propusemos avaliar o perfis imunofenotípico (quanto à expressão de CD34 e CD133) por citometria de fluxo e de expressão gênica de células CD34+ e células CD133+ selecionadas imunomagneticamente, de ambas as fontes, pelas técnicas de microarray e PCR em tempo real. Nossos resultados revelaram que enquanto a maioria das células CD133+ são CD34+, independente da fonte, as células CD34+ de SCU possuem uma porcentagem significativamente maior de células CD133+ do que às células CD34+ de MO. A análise de clusterização revelou que as células CD133+ de MO se agrupam com as células de SCU (CD34+ e CD133+), enquanto as CD34+ de MO aparecem como um grupo distinto. A comparação dos perfis de expressão gênica entre as células CD133+ e as células CD34+, revelou a hiper-expressão... (Resumo completo, clicar acesso eletrônico abaixo) Abstract: A higher expression of transcription targets and components of the NF-kB pathway is a distinctive feature of umbilical cord blood (UCB) CD34+ hematopoietic stem cells (HSC) when compared to bone marrow (BM) CD34+ HSC and this could be related to the more primitive state of the newborn's HSC. However, CD34+ cells represent a heterogeneous group of cells composed by stem and progenitors cells in different developmental stages, and differences in cellular composition between both sources could contribute for these finding. The surface marker CD133 has been identified as a very primitive marker, expressed in a subpopulation of CD34bright, with a proposal hemangioblast potential. Thus, in attempt to better characterize the cellular composition of UCB and BM and to identify molecular mechanisms related to the more primitive characteristics of CD133+ cells, we proposed to evaluate the immunophenotypic profile (expression of CD34 and CD133) by flow-cytometry and the gene expression profiles of immunomagnetically selected CD34+ and CD133+ cells, from both sources, by microarray and Real time PCR. Our results highlighted that, while almost all CD133+ cells are CD34+ independently of the evaluated source, the UCB CD34+ cells showed a significantly higher proportion of CD133 expression, compared to BM CD34+ cells. After obtaining the expression profiles from distinct HSC pooled samples generated by microarrays, cluster analysis showed that BM CD133+ cells preferentially grouped with UCB cells (CD34+ and CD133+) instead of BM CD34+ cells, which appeared as a very distinct profile The comparison between CD133+ and CD34+ samples revealed the over-expression of 47 transcriptional factors (TF) in CD133+ cells, many of them well-known and related... (Complete abstract click electronic access below) Mestre

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Dubory, Arnaud. "Interface os-implant titane et ingénierie tissulaire A cadaveric validation of a method based on impact analysis to monitor the femoral stem insertion Bone marrow mesenchymal stem cells predict radiographic osseointegration of cementeless titanium hip cups Survival, adhesion, and expression of osteoblastic genes of human derived-bone marrow mesenchymal stromal cells on PEEK and titanium-coated PEEK lumbar interbody cages." Thesis, Paris Est, 2018. http://www.theses.fr/2018PESC0067.

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Introduction : Le titane, à la fois utilisé comme moyen de fixation d’un implant devant ses propriétés biomécaniques proches de l’os et comme moyen d’ostéointégration a pris une place prépondérante en orthopédie. L’ostéointégration d’un implant titane (IT) essentielle pour la pérennité de ce dernier, dépend de 2 phases : une phase primaire MECANIQUE correspondant à l’impaction ou tenue primaire de l’IT et une phase secondaire BIOLOGIQUE correspondant à la colonisation de l’IT par le tissu osseux.L’objectif de ce travail fut d’évaluer et d’améliorer lors de ces deux phases la stabilité et l’ostéointégration de l’implant titane :(1) Évaluer la tenue primaire des tiges fémorales en titane non cimentées par l’analyse de l’impact correspondant à la mesure de l’impact au cours du temps.(2) Évaluer si la quantité de CSM contenues dans l’aile iliaque est corrélée à la survie sans reprise de des implants acétabulaires impactés dans le cadre de l’ostéonécrose aseptique de la tête fémorale.(3) Améliorer l’ostéointégration des IT par les méthodes de thérapie cellulaire en étudiant in vitro la survie et la division des cellules stromales mésenchymateuses humaines (CSM) osseuses au contact de cages lombaires inter-somatiques recouvertes d’alliage titane rugueux.Matériels et Méthodes: L’évaluation de la tenue primaire des tiges fémorales en titane sans ciment selon l’analyse de l’impact a été réalisée à l’aide d’un marteau muni d’un capteur de force piézo-électrique sur 20 sujets anatomiques soit 40 hanches. On a comparé le nombre de coups de marteau pour obtenir l’impaction idéale de la prothèse selon 3 méthodes d’évaluation différentes : le nombre de coups nécessaires pour le chirurgien (Nchir), le nombre de coups nécessaires par l’analyse vidéo de l’enfoncement de la tige dans le fémur (Nvid) et le nombre de coups nécessaires par l’analyse de l’impact (NI).Pour savoir si la quantité de CSMs dans la crête iliaque pouvait refléter l’ostéointégration des implants acétabulaires impactées et le risque de reprise chirurgicale, on a comparé le taux de CSMs mesuré lors de la réalisation d’une ponction concentration et réinjection dans la zone d’ostéonécrose et l’évolution clinico-radiographique des implants acétabulaires mis en place par la suite pour ces même patients (n=90) qui ont eu une arthroplastie totale de hanche in fine. Le recul moyen était de 15 ans.La survie cellulaire des CSM osseuses humaines a été évaluée sur des cages intersomatiques lombaires recouvertes de titane. Trois groupes (n=5) furent constitués : un groupe contrôle, un groupe cages avec surface titane, un groupe cage sans titane. Sur chaque implant, 1 microlitre contenant 106 CSM osseuses humaines a été mis en culture. L’analyse de la survie cellulaire, de la prolifération cellulaire et de l’expression de gènes de différenciation ostéoblastique ont été réalisés et comparés.Résultats : Concernant la première étude sur l’intérêt de l’analyse de l’impact pour la tenue de la tige fémorale, la différence entre NI, Nchir et Nvid était inférieure ou égale à 3 pour plus de 85% des configurations réalisées. Concernant la deuxième étude, il a été mis en évidence qu’un faible nombre de CSM dans la crête iliaque était un facteur de risque de révision chirurgicale chez les patients traités par un implant acétabulaire sans ciment. La troisième étude a montré que les CSMs pouvaient survivre, se développer pendant 96 heures et exprimer des gènes de différenciation ostéoblastique de manière identique sur les cages avec ou sans titane.Conclusion : L’analyse de l’impact permet de donner une information objective sur la tenue primaire de la tige fémorale en titane et impactée. Le titane est aussi un milieu favorisé de survie et de prolifération des CSM osseuses prédestinées à devenir des cellules ostéoformatrices surtout qu’un faible nombre de CSM semble être un risque d’échec d’ostéointégration des implants acétabulaires sans ciment Introduction: Titanium, both used as a mean of fixing an implant to its biomechanical properties close to the bone and as a mean of osseointegration has taken a prominent place. The implant stability is essential for the durability of a titanium implant (TI); it depends on 2 phases: a primary phase, MECHANICAL, corresponding to the impaction or primary holding of the TI and a secondary phase, BIOLOGICAL, corresponding to the colonization of TI by bone tissue.The objective of this work was to evaluate and improve during these two phases the osseointegration of the titanium implant:(1) To evaluate the primary stability of uncemented titanium femoral stems by impact analysis corresponding to the measurement of impact over time.(2) To evaluate whether the amount of mesenchymal stromal cells (MSCs) contained in the iliac crest is correlated with the non-recovery survival of acetabular implants impacted in a context of aseptic osteonecrosis of the femoral head.(3) To improve the osseointegration of TI by cell therapy methods in vitro by studying the survival and division of human MSCs in contact with interbody lumbar cages coated with rough titanium alloy.Methods: The evaluation of the primary stability of cementless titanium femoral stems according to the impact analysis was carried out using a hammer equipped with a piezoelectric force sensor on 20 anatomical subjects, i.e. 40 hips. The number of hammer strokes was compared to obtain the ideal impaction of the prosthesis according to 3 different evaluation methods: number of impacts required by the surgeon (Nsurg), number of impacts required by the video analysis of the depression of the stem in the femur (Nvid), numbers of impacts needed by the impact analysis (Ni).To determine whether the amount of MSCs in the iliac crest could reflect the osseointegration of impacted acetabular implants and the risk of surgical revision. The rate of MSCs measured when performing a surgical cell therapy for aseptic osteonecrosis of the femoral head and the clinical and radiographic outcome of acetabular implants subsequently established for these same patients (n = 90), who had total hip arthroplasty in fine were compared. The mean follow-up was 15 years.The cell survival of bone marrow-derived MSCs was evaluated on lumbar interbody cages coated with titanium. Three groups (n = 5) were formed: a control group, a cage group with titanium surface, a cage group without titanium. On each implant, 1 microliter containing 106 human bone MSCs was cultured. The analysis of cell survival, cell proliferation and expression of osteoblastic genes were performed and compared.Results: Regarding the impact analysis of the cementless femoral stem impaction, the difference between NI, Nchir and Nvid was lower than 3 for more than 85% of the configurations performed.For the second study, a small number of MSCs in the iliac crest was a risk factor for surgical revision in patients treated with a cementless acetabular implant.The third study showed that MSCs could grow until 96 hours and could express osteoblastics genes 21 days after cell seed. No difference between PEEK cage and Titanium-coated PEEK has been found.Conclusion: The impact analysis provides objective data on the primary holding of the titanium impacted femoral stem. Titanium is also a favored biomaterial for the survival and proliferation of bone marrow-derived MSC predestined to become osteforming cells, especially since a small number of MSCs seems to be a risk of failure of osseointegration of cementless acetabular implants

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Qadan, Maha Ahmad. "Sourcing and Modulation of the Fate of Connective Tissue Progenitors." Kent State University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=kent1479416651140376.

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Milani, Cintia. "Expressão gênica diferencial das células estromais obtidas de medula óssea na presença ou ausência de célula tumoral oculta em pacientes com câncer de mama." Universidade de São Paulo, 2006. http://www.teses.usp.br/teses/disponiveis/5/5155/tde-19032010-115152/.

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A célula estromal pode influenciar o desenvolvimento do tumor no sítio primário e secundário, mas pouco é conhecido sobre as características moleculares das células estromais presentes na medula óssea de pacientes com câncer de mama. Nosso objetivo foi avaliar a expressão gênica diferencial entre as células estromais oriundas de medula óssea na presença ou ausência de célula tumoral oculta. Coletamos dez aspirados de medula óssea das pacientes com câncer de mama. A classificação do comprometimento da medula por células tumorais ocultas foi realizada pela detecção da expressão de CK19 por Nested-RT-PCR e quatro entre dez pacientes apresentaram presença de célula tumoral na medula óssea. Estabelecemos culturas primárias de células estromais de todas as amostras e, selecionamos amostras originárias de duas pacientes contendo linfonodos comprometidos e presença de célula tumoral oculta em medula e também de duas pacientes que não apresentavam linfonodos comprometidos e nem célula tumoral oculta na medula. As pacientes selecionadas eram pós-menopausadas com diagnóstico de carcinoma ductal invasor e expressão imunohistoquímica positiva para receptor de estrógeno e progesterona. Realizamos avaliação do perfil de expressão gênica entre estes dois grupos, o que nos revelou 21 genes diferencialmente expressos dentre os 4.608 genes imobilizados em lâmina de cDNA microarray; nove genes hiperexpressos em célula estromal de medula comprometida (PTHLH, TLOC1, NCOA6, C17orf57, ANAPC11, MAST4, POLR3E, CPNE1 e B4GALT5) e doze genes hipoexpressos em célula estromal de medula comprometida (MRPL2, NAT10, DAP, RNF2, FLOT2, FKBP10, SLIT3, EBNA1BP2, SLC35B2, MICAL2, GPR3, TSPAN17). Nossos dados sugerem que apesar da expressão gênica de células estromais oriundas de medula óssea comprometida ou não por micrometástases ser semelhante, algumas diferenças podem ser identificadas. Stromal cells may influence tumor development in primary and secundary sites, however, molecular characteristics of bone marrow stromal cells from breast cancer patients are almost unknown. Our aim was to evaluate the differential gene expression of bone marrow stromal cells from breast cancer patients in the presence or abscence of occult tumor cells. Bone marrow (BM) aspirates were obtained from 10 breast cancer patients. The presence of occult bone marrow disseminated tumor cells was detected by CK19 expression quantified by reverse transcriptase polymerase chain reaction (RT-PCR). Presence of tumoral cell was detected in four of ten BM samples. Stromal cells primary cultures were established and samples from two patients with positive lymph nodes and presence of occult tumor cells in bone marrow and samples from two patients with negative lymph nodes and abscence of occult tumor cells in bone marrow were selected. All the included patients were postmenopausal with invasive ductal carcinoma and positive estrogen and progesterone receptors detected by immunohistochemical analysis. Gene profile evaluated in cDNA microarray slides containing 4.608 spotted genes revealed 21 differencially expressed genes, nine upregulated (PTHLH, TLOC1, NCOA6, C17orf57, ANAPC11, MAST4, POLR3E, CPNE1 e B4GALT5) and twelve downregulated (MRPL2, NAT10, DAP, RNF2, FLOT2, FKBP10, SLIT3, EBNA1BP2, SLC35B2, MICAL2, GPR3, TSPAN17) in stromal cell derived from bone marrow in the presence of tumor breast cancer cell. Our data suggest that gene expression from bone marrow derived stromall cells in the presence or abscence of occult tumor cells seems similar, however small differences may be identified.

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Valle, Paulo Roberto Del. "Perfil transcricional de fibroblastos de tumor primário, linfonodo e medula óssea de pacientes com câncer de mama." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/5/5155/tde-26032013-143422/.

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Introdução: Em câncer de mama, existem evidências de que o microambiente pode influenciar o desenvolvimento do tumor no sítio primário, bem como em metástases regionais e a distância. Neste contexto, fibroblastos são importantes células estromais que podem influenciar a proliferação e a migração de células do câncer e podem prover um nicho apropriado para o desenvolvimento tumoral. Objetivos:O principal objetivo deste trabalho é comparar células estromais obtidas do tumor primário (PT), metástase linfonodal (N+) e medula óssea (BM) de pacientes com câncer de mama, através do perfil de expressão gênica. Pacientes e Métodos: Foi analisada a expressão gênica de fibroblastos (cultura primária) de 11 pacientes com câncer de mama. O perfil de expressão foi determinado em PT (n=4), N+(n=3) e BM (n=4) através de uma plataforma de cDNA microarray customizada (contendo 4.800 sequencias imobilizadas, representando cerca de 4600 genes), e os genes diferencialmente expressos foram identificados pelo teste SAM multiclasse, seguido pelo teste SAM de duas classes (TMEV, FDR 0%). A análise funcional foi realizada pelo software DAVID v6.7. Validação técnica foi realizada em 6 amostras previamente analisadas no microarray e a validação biológica em fibroblastos obtidos de outros 16 pacientes utilizando-se de RT-qPCR. Resultados: O perfil de expressão gênica dos fibroblastos obtidos de diferentes sítios mostraram 267 genes diferencialmente expressos, os quais apropriadamente agruparam os fibroblastos de acordo com suas origens (PT vs. N+ vs. BM). Apesar das diferenças entre PT e N+ serem representadas por 20 genes, as diferenças entre PT vs. BM e N+ vs BM foram mais significantes (235 e 245 genes diferencialmente expressos respectivamente). Análise funcional dos genes diferencialmente expressos mostrou enriquecimento de funções relacionadas ao desenvolvimento e morfogênese.A seguir, a expressão de alguns genes selecionados foi analisada em uma série diferente de amostras (validação biológica). Desse modo observamos que NOTCH2 confirmou uma alta expressão em N+ (vs. PT), e ADCY2, HECTD1, HNMT, LOX, MACF1 e USP16 confirmaram alta expressão em BM (vs PT). Conclusão:Em pacientes com câncer de mama, células estromais obtidas de diferentes origens apresentam um perfil de expressão gênica diferencial, o qual pode influenciar o comportamento do tumor may influence tumor development in the primary site of breast cancer, as well as in regional and distant metastatic sites. In this context, fibroblasts are important stromal cells which influence proliferation and migration of cancer cells and may also provide an appropriate niche to tumor development. Objectives: The main objective of this work is the comparison of stromal cells from the primary tumor (PT), lymph node metastasis (N+) and bone marrow (BM) obtained from breast cancer patients, through gene expression profile. Patients and Methods: The gene expression profile was analyzed in fibroblasts primary culture from 11 breast cancer patients. The expression profiles of PT cells (n=4), N+ cells (n=3) and BM cells (n=4) were determined through a customized cDNA microarray platform (containing 4800 immobilized sequences which represents 4600 genes approximately). The analysis were performed by SAM multiclass (TMEV; FDR 0%), followed by SAM two classes test (TMEV; FDR 0%). Functional analysis was performed using DAVID v6.7. Technical validation was performed in same 6 samples that were previously analyzed in microarray experiments and biological validation was performed in fibroblasts obtained from other group of 16patients by RT-qPCR Results: The expression profile of fibroblasts obtained from three sites revealed 267 differentially expressed genes, which appropriately clustered fibroblasts in three different branches, in accordance with their origin (PT vs. N+ vs. BM). Although the differences between PT and N+ were represented by 20 genes, differences between PT vs. BM and N+ vs. BM were more significant (235 and 245 differentially expressed genes respectively). Functional analysis revealed enrichment of functions related to development and morphogenesis. Afterwards, the expression of some selected genes were analyzed in a different batch of samples (biological validation).Thereby, NOTCH2 confirmed high expression in N+ (vs. PT), and ADCY2, HECTD1, HNMT, LOX, MACF1 and USP16 confirmed high expression in BM (vs. PT). Conclusion: In breast cancer patients, stromal cells obtained from different origins present a differential gene expression profile, which may influence tumor behavior

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Hoflack, Bernard, Pierre Jurdic, Thilo Riedl, Anne Gallois, and Maria Arantzazu Sanchez-Fernandez. "Osteoclasts control osteoblast chemotaxis via PDGF-BB/PDGF receptor beta signaling." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-184120.

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BACKGROUND: Bone remodeling relies on the tightly regulated interplay between bone forming osteoblasts and bone digesting osteoclasts. Several studies have now described the molecular mechanisms by which osteoblasts control osteoclastogenesis and bone degradation. It is currently unclear whether osteoclasts can influence bone rebuilding. METHODOLOGY/PRINCIPAL FINDINGS: Using in vitro cell systems, we show here that mature osteoclasts, but not their precursors, secrete chemotactic factors recognized by both mature osteoblasts and their precursors. Several growth factors whose expression is upregulated during osteoclastogenesis were identified by DNA microarrays as candidates mediating osteoblast chemotaxis. Our subsequent functional analyses demonstrate that mature osteoclasts, whose platelet-derived growth factor bb (PDGF-bb) expression is reduced by siRNAs, exhibit a reduced capability of attracting osteoblasts. Conversely, osteoblasts whose platelet-derived growth factor receptor beta (PDGFR-beta) expression is reduced by siRNAs exhibit a lower capability of responding to chemotactic factors secreted by osteoclasts. CONCLUSIONS/SIGNIFICANCE: We conclude that, in vitro mature osteoclasts control osteoblast chemotaxis via PDGF-bb/PDGFR-beta signaling. This may provide one key mechanism by which osteoclasts control bone formation in vivo.

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Fioroni, Orietta Maria. "Gene expression in cultured cells." Thesis, University of Leicester, 1989. http://hdl.handle.net/2381/35455.

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Dedifferentiation is the process by which specialised quiescent cells give rise to heterotrophic, dividing cells. This process may be initiated in vivo as a response to wounding, or in vitro during culture initiation. This thesis is concerned with evaluating whether the process of dedifferentiation and maintenance of the fast-dividing, dedifferentiated state by culture, is dependant upon major changes in gene expression. In particular, the role of transcription, as mirrored by changes in steady state mRNA levels, in these putative changes in gene expression has been investigated. Mechanically isolated Asparagus officinalis mesophyll cells were used to study dedifferentiating cells, and suspension cultures of Petunia hybrida to investigate the established dedifferentiated state. This thesis shows that dedifferentiation in Asparagus officinalis is accompanied by major changes in the steady state mRNA profiles of the cells. A group of novel transcripts appearing in dedifferentiating asparagus cells were termed DDl, and targeted for further study. Two cDNA clones coding for DDl transcripts were isolated and characterised, and antibodies to DDl raised for serological work. Only minor differences were found between the steady state mRNA populations of Petunia hybrida cultured cells and seedlings, and these were mainly caused by transcripts disappearing in culture; no transcripts specific to the suspension culture system were detected. The results presented in this thesis are used to foward the hypothesis that changes in gene expression involving de novo transcription may only occur in response to major changes in environmental conditions. It is suggested that the basal transcription pattern for cells in established state is probably common to all cell types with regards to primary cell functions such as growth, division and catabolism. In such established states, the control of metabolism probably resides within the biochemical pathways utilised by the cell at any moment in time.

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Hoflack, Bernard, Pierre Jurdic, Thilo Riedl, Anne Gallois, and Maria Arantzazu Sanchez-Fernandez. "Osteoclasts control osteoblast chemotaxis via PDGF-BB/PDGF receptor beta signaling." PLOS one, 2008. https://tud.qucosa.de/id/qucosa%3A28994.

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BACKGROUND: Bone remodeling relies on the tightly regulated interplay between bone forming osteoblasts and bone digesting osteoclasts. Several studies have now described the molecular mechanisms by which osteoblasts control osteoclastogenesis and bone degradation. It is currently unclear whether osteoclasts can influence bone rebuilding. METHODOLOGY/PRINCIPAL FINDINGS: Using in vitro cell systems, we show here that mature osteoclasts, but not their precursors, secrete chemotactic factors recognized by both mature osteoblasts and their precursors. Several growth factors whose expression is upregulated during osteoclastogenesis were identified by DNA microarrays as candidates mediating osteoblast chemotaxis. Our subsequent functional analyses demonstrate that mature osteoclasts, whose platelet-derived growth factor bb (PDGF-bb) expression is reduced by siRNAs, exhibit a reduced capability of attracting osteoblasts. Conversely, osteoblasts whose platelet-derived growth factor receptor beta (PDGFR-beta) expression is reduced by siRNAs exhibit a lower capability of responding to chemotactic factors secreted by osteoclasts. CONCLUSIONS/SIGNIFICANCE: We conclude that, in vitro mature osteoclasts control osteoblast chemotaxis via PDGF-bb/PDGFR-beta signaling. This may provide one key mechanism by which osteoclasts control bone formation in vivo.

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Zachos, Terri A. "Gene-augmented mesenchymal stem cells in bone repair." Columbus, Ohio : Ohio State University, 2006. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1146076285.

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Moudgil, Suniti 1976. "Organosilicate nanoparticles as gene delivery vehicles for bone cells." Thesis, Massachusetts Institute of Technology, 2004. http://hdl.handle.net/1721.1/28307.

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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2004. Includes bibliographical references. (cont.) proliferation. The metabolic response of these cells after particle ingestion was also characterized in order to ensure that the osteoblasts retained their phenotype. The expression of various proteins involved in bone formation, such as alkaline phosphatase, osteocalcin, osteopontin and fibronectin was quantified. The results indicated that osteoblasts retained their phenotype after organosilicate nanoparticle ingestion. The levels of various cytokines expressed during inflammatory response remained low due to the biocompatibility of amorphous silica. An optimized Ca-SiO₂ nanoparticulate system was developed with maximum particle uptake that enhanced cell viability. A model gene delivery system was created by complexing these nanoparticles with plasmid DNA that encoded for green fluorescent protein (GFP). The effects of nanoparticle size, composition and surface charge on complex size, DNA binding affinity and subsequent GFP expression in osteoblasts were investigated in detail. In addition to primary and immortalized osteoblasts, we have studied the effect of this gene delivery system on two other control cell lines: fibroblasts (connective tissue cells) and hepatocytes (non-connective tissue cells). The Ca-SiO₂-DNA complexes displayed significantly higher transfection efficiencies in osteoblasts and fibroblasts relative to hepatocytes compared to lipofectamine-DNA complexes. In addition, Ca-SiO₂-DNA complexes enhanced osteoblast cell proliferation while achieving successful transfection ... While bone has a substantial capacity to heal itself, there are approximately 1 million fractures that occur in the U.S. annually that are difficult to heal. These include fractures that occur at sites of low vascularity, fractures that result in a large amount of tissue loss, and fractures that result from bone fragility syndromes such as osteoporosis. There has been a great deal of interest in the tissue engineering of bone in order to treat such fractures. One major aspect of the tissue engineering approach involves the addition of growth factors or proteins to synthetic grafts to accelerate bone regeneration. However, delivering these proteins at the appropriate times and therapeutic levels poses significant challenges. Alternatively, delivering the genes that encode for these proteins could offer a more effective treatment, since proper incorporation of the appropriate genes into cellular nuclei would allow the cells to manufacture authentic protein products. The motivation of this research was to design new materials for gene delivery to bone cells. Conventional non-viral vectors are plagued by toxicity and low transfection efficiencies. The purpose of this work was to design bioactive nanoparticles that could enter the osteoblast membrane without inducing toxicity. These materials were silicate-based, since doped silicates have been shown to possess osteogenic properties. A method to synthesize monodisperse, spherical organosilicate nanoparticles using a surfactant-stabilized sol-gel technique was developed. Surface dopants such as Ca, Mg and Na were found to influence cellular response to nanoparticles. In addition to particle composition, particle size was also found to have a significant effect on osteoblast uptake and cell by Suniti Moudgil. Ph.D.

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Pardo, Steven Javier. "Effects of simulated microgravity on preosteoblast gene expression." Thesis, Available online, Georgia Institute of Technology, 2004:, 2004. http://etd.gatech.edu/theses/available/etd-06072004-131314/unrestricted/pardo%5Fsteven%5Fj%5F200405%5Fms.pdf.

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Singhatanadgit, Weerachai. "Expression and regulation of bone morphogenetic protein receptors in human alveolar bone cells." Thesis, University College London (University of London), 2007. http://discovery.ucl.ac.uk/1445851/.

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Bone morphogenetic proteins (BMPs) are members of the transforming growth factor P (TGF-P) superfamily of growth factors that stimulate osteoblast differentiation and function. They exert their biological activities through signal transduction via three specific serine/threonine kinase transmembrane receptors, designated types-IA, -IB and -II (BMPR-IA, -IB and -II). These BMP-specific receptors thus determine in part the sensitivity and responsiveness of target cells to the BMP and thereby the biological activity of the BMP. However, little is known about BMPR-IA, -IB and -II in bone. The present study was therefore carried out to examine the expression and regulation of the BMPR in primary human alveolar bone (AB) cells in vitro. Cells were obtained from explants of human AB and exhibited a number of characteristic phenotypic features of osteoblasts. They were also found to express all three BMPR mRNA transcripts and proteins, each of which had a unique subcellular distribution. For example, in addition to its expected localisation at the plasma membrane, a major proportion of BMPR-IB was also observed in the cytoplasm and the nucleus. Moreover, the distribution of BMPR-IB was found to be highly regulated by TGF-pi, which caused a pronounced translocation of this receptor to the plasma membrane, resulting in a marked increase in BMP-2 binding and bone cell response. The BMPR also appeared to be differentially controlled at the post-translational level by inflammatory cytokines, which were shown, for the first time, to cause shedding of the cell surface proteins and the concurrent generation of 'soluble' forms of the BMPR. IL-1 p and TNF-ct were found to significantly induce the shedding of soluble BMPR-IB specifically, thereby reducing BMPR-IB surface expression and diminishing BMP-2- induced AB cell functions, such as Smad 1/5/8 phosphorylation, alkaline phosphatase (ALP) activity and osteocalcin (OC) expression. In contrast, the expression of BMPR-IB was found to be up-regulated by osteogenic growth factors including TGF-pl, FGF-2 and PDGF-AB, which enhanced BMP-2-induced AB cell functions. The biological importance of BMPR-IB in these cells was established using an RNA interference approach, which demonstrated that the expression of pivotal osteoblast-associated genes ALP, OC, distal-less homeobox 5 (Dlx5) and core binding factor alpha1 (Cbfalpha1) was dependent on the BMPR-IB signalling pathway. In conclusion, the activities of the BMPR-IA, -IB and -II genes in primary human AB cells were found to be controlled by a number of biological mediators. In addition, the expression of these receptors was also regulated at both the transcriptional and post- translational levels, with BMPR-IB being the most responsive receptor, at least in vitro. These findings suggest that BMPR-IB could thus be a possible therapeutic target for eliciting improved BMP-induced bone healing in vivo.

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Quinn, Michael Corwin James, and n/a. "Gene expression profiling of human granulosa cells." University of Otago. Department of Anatomy & Structural Biology, 2005. http://adt.otago.ac.nz./public/adt-NZDU20070501.144002.

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Human granulosa cells play an important role in the follicle, providing the oocyte with nutrients and growth factors to ensure successful ovulation. Normal granulosa cell functioning is thus crucial to human fertility. By studying their transcriptome, the mechanisms underpinning follicle development and infertility will be better understood. In this study, granulosa cells were retrieved at the time of oocyte removal for in vitro fertilization (IVF). Cells were purified through a combination of a Percoll gradient to remove red blood cells and positive selection of granulosa cell aggregations. On average, contamination by white blood cells was 2% as measured by FACS analysis using specific white blood cell markers. Histological and electron microscopy of granulosa cell aggregations did not detect evidence of resident ovarian white blood cells. This technique provided a good source of pure, healthy granulosa cells for RNA extraction and subsequent gene expression studies. The construction of a human granulosa cell SAGE library derived 1689 SAGEtags and 1289 discrete mRNA transcripts. SAGEtags for a number of well recognized granulosa cell genes (FSH receptor, follistatin, connexin 43) were found in addition to hormone receptors, multiple kinases, structural genes, apoptosis related genes and secreted proteins. A variety of other SAGE libraries were downloaded from SAGEmap (two ovary epithelium, ovary, white blood cell, brain, cerebellum, heart, liver, lung, kidney, pancreas and universal human reference) and compared to the human granulosa cell library. This was based on two measures: a gene specificity score (GSS) and a tissue specificity score (TSS). Three SAGEtags were identified with high levels of expression in granulosa cells, but no or low expression in the other libraries. These were retinol binding protein 1 (RBP 1), scavenger receptor class B member 1 (SCARB 1) and hydroxysteroid (11-beta) dehydrogenase 1 (11-β-HSD). Library comparisons were validated by real time RT-PCR. The TSS score revealed granulosa cells were most similar to the universal reference library and least similar to liver. Granulosa cell samples were collected from woman undergoing IVF for a range of infertility disorders. These included polycystic ovary syndrome (PCOS), tubal disease, endometriosis and idiopathic (unknown). Gene expression was compared between these groups using real time RT-PCR. Candidate genes included RBP 1, 11-β-HSD, SCARB 1, FSH receptor, follistatin, decidual protein induced by progesterone, and progesterone membrane receptor component 1 and 2. Granulosa cell gene expression was significantly different (p<0.05) from human white blood cells. No differences were found in gene expression levels between the infertility disorders. Analysis of each patient, however, revealed individuals with marked over-expression of selected genes. The technique of Generation of Longer cDNA fragments for Gene Identification (GLGI) was used to investigate seven SAGEtags that did match known genes or ESTs. Although no novel genes were characterized, a further 14 granulosa cell transcripts were identified by this technique. This thesis used an integrated approach to the study of human granulosa cell gene expression. This has involved development of a purification method, the use of a high-throughput technique (SAGE), bioinformatics tools to identify candidates genes, real time RT-PCR to investigate gene expression of particular genes in infertility disorders and finally a technique with the potential to characterize unknown SAGEtags (GLGI). This systematic approach has advanced the understanding of gene expression in human granulosa cells and identified avenues for future research into folliculogenesis and human infertility.

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40

Holder, Emma L. (Emma Lesley). "Gene expression in muscle tissue and cells." Thesis, McGill University, 1993. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=69755.

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Cellular differentiation is accompanied by the modulation of gene expression. I have compared the expression of various genes, using the slot-blot technique, in two different systems. First, the level of expression of a wide variety of genes was analyzed in the hypertrophied heart of transgenic mice expressing the polyomavirus large T-antigen gene, and compared to normal control heart. I have shown that most changes in gene expression occurred mainly during early stages of heart hypertrophy. These genes code for proteins known to play a role in signal transduction, and transcriptional and growth control. The latter stages of cardiac hypertrophy are accompanied by changes in the expression of genes that are mostly involved in stress responses. Second, we analyzed the expression of various genes in three mouse myogenic cell lines undergoing differentiation in several culture conditions. The adult (C2C12) and fetal-derived (G7 and G8) myoblast cell lines were exposed to either retinoic acid, dimethyl sulfoxide, or transforming growth factor $ beta$. These three molecules are known to have profound effects on cellular growth and differentiation. I have shown that these treatments result in significant changes in expression of a wide variety of genes. Interestingly, all three cell lines differed considerably in their pattern of gene expression. Results from the analysis of these two systems demonstrate that differentially induced morphological changes of muscle cells, result in cell type specific changes in expression of a variety of genes.

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41

Bahn, S. "Directing gene expression to cerebellar granule cells." Thesis, University of Cambridge, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.596247.

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The aim of this thesis was to investigate mechanisms involved in activating gene expression in a distinct neuronal sub-population. As a model system, I have chosen the GABAA receptor α6 subunit gene. Within the whole brain, α6 gene expression is restricted to differentiated cereballar granule cells and the lineage-related granule cells in the cochlear nucleus. I searched for regulatory elements using constructs derived from regions within the α6 gene in a transgenic mouse assay, linking these regions to the β-galactosidase reporter gene to assay for transcriptional activity. A 7.2 kb DNA fragment containing a 1 kb portion upstream of the start site(s), together with exons 1-8, directs cerebellar granule cell-specific reporter gene expression. Truncated versions of this 7.2 kb transgene were analysed; none of these smaller transgenes gave consistent granule cell-specific expression. As an adjunct to identify cerebellar granule cell-specific DNA elements, a comparative approach was employed. If the expression pattern of the α6 gene is conserved in evolutionary distant species, then sequences involved in spatially regulating this gene expression may also be conserved. I cloned α6 cDNA homologues in distant species (goldfish and chicken) and showed that α6 expression was restricted to cerebellar granule cells. Comparative genome sequencing was used to look for conserved DNA regions outside the coding region of the α6 gene. The chicken and pufferfish α6 gene structures were mapped and the corresponding regions of the mouse, chicken and pufferfish genomic clones were sequenced. Two small regions of sequence identity were identified in the 5' proximal area in all three species. These regions could be binding sites for transcription factors. Cerebellar granule cell precursors can switch on the α6 gene ectopically. This implies that α6 gene expression is cell-intrinsically regulated.

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Assidi, Mourad. "Oocyte competence and cumulus cells gene expression." Thesis, Université Laval, 2010. http://www.theses.ulaval.ca/2010/27679/27679.pdf.

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Dickinson, P. "Fibronectin gene expression in higher eukaryotic cells." Thesis, University of Manchester, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.378322.

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Watson, Christine J. "Gene expression in murine embryonal carcinoma cells." Thesis, Imperial College London, 1986. http://hdl.handle.net/10044/1/38181.

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Lafta, Inam Jasim. "STAG3 gene expression in breast cancer cells." Thesis, University of Sheffield, 2016. http://etheses.whiterose.ac.uk/12329/.

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The expression of cohesin genes has been found to be disregulated in a number of cancers including prostate, breast and squamous cell carcinoma; and mutations in genes that encode cohesin components have been noticed in colorectal cancer and myeloid malignancies (reviewed by Rhodes et al., 2011). It has been suggested that members of the cohesin complex might be considered as a subgroup of cancer biomarkers (Xu et al., 2011a). Therefore, this study focused on studying the expression of STAG genes in breast cancer cell lines and primary breast tumours. More attention was paid for STAG3, because in addition of being the meiotic component of cohesin is also a member of Cancer Testis (CT) antigens. CT antigens have been used successfully as cancer biomarkers as well as therapeutic targets for various malignancies. Our findings show that the STAG1, STAG2 and STAG3 genes are highly expressed at the mRNA level in the breast cancer cell lines including: MCF-7, T-47D, MDA-MB-231 and MDA-MB-468 and primary breast tumours as well compared to normal breast tissue or normal breast cell line, MCF-10A, using qRT-PCR. Interestingly, a tendency for increasing STAG3 mRNA expression was recorded from stage I through stage IV of breast tumour implying that there might be increased expression as the tumour develops. Therefore, STAG3 expression was confirmed at the protein level by immunoblotting, where STAG3 protein bands were produced by all of the studied cancer cells when compared with the normal breast. Jurkat cells were used as a positive control as STAG3 expression in these had been previously established. Further confirmation of STAG3 protein signal was achieved in primary breast tumour tissue sections compared with the normal tissue using immunohistochemistry. Overall, these data suggest that STAG3 may be a suitable novel biomarker for breast cancer detection. Because STAG3 is a potential therapeutic target for breast cancer, RNA interference was successfully used to deplete STAG3 in MCF-7 cells. Analysis of the cell cycle profile by FACS revealed an accumulation of cells in G1 phase, and simultaneous reduction in the number of cells in both S and G2/M phases of the cell cycle. However, when depleting STAG3 using other si-RNAs specific for STAG3, more breast cancer dead cells were reported in MTT toxicity assay compared to MCF-10A. Finally, we studied STAG3 regulation by the transcription factors, E2F4/E2F6, no correlation was found between STAG3 expression and either of E2Fs as depleting any of them did not affect STAG3 expression. Interestingly, we found that RNAi-mediated E2F6 silencing, but not E2F4, in cancer cells caused cell death. On the other hand, MCF-10A cells depleted of E2F6 showed higher survival fraction in MTT. This finding suggests E2F6 as another potential therapeutic target for breast cancer.

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46

Ruchala, Monika. "Mitochondrial Gene Expression in Human Mononuclear Cells." VCU Scholars Compass, 2014. http://scholarscompass.vcu.edu/etd/3530.

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MITOCHONDRIAL GENE EXPRESSION IN HUMAN MONONUCLEAR CELLS By Monika D. Ruchała, M.S. A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science at Virginia Commonwealth University. Virginia Commonwealth University, 2014. Director: Dr. James P. Bennett Jr, M.D., Ph.D., Bemiss Professor Departments of Neurology, Psychiatry and Physiology and Biophysics Adult neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), have been intensively studied in recent years in pursuit of mechanisms responsible for origin and progression. One emerging theme is mitochondrial energetic deficiency as a mechanism of neuronal death. Recent descriptions of protocols to generate induced pluripotent stems cells (iPSCs) from living patients offer the potential to create unique disease models. This model can potentially lead to crucial advances in developing treatment options for a wide variety of neurodegenerative diseases. In this thesis, we attempt to induce iPSCs from mononuclear cells (MNC) in peripheral blood acquired from patients with ALS and healthy control (CTL) subjects, and analyze their mitochondrial genomes. The reprogramming of MNC to yield iPSC was done by nucleofection of an episomal plasmid pEB C5, expressing OriP sequences of the EpsteinBarr and five reprogramming transgenes Oct4, Sox2, Klf4, cMyc and Lin28. We investigated the expression of mitochondrial DNA genes, ND2, ND4, COXIII and 12s rRNA in the ALS and CTL MNC before and after their culturing. The results implicate deregulated mitochondrial bioenergetics as a characteristic of ALS. Future work will establish whether these abnormalities in mitochondrial bioenergetics persist in iPSC’s and iPSC-derived neurons from ALS subject

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47

Allay, James Andre. "Retroviral-mediated gene transduction of bone marrow-derived stem cells." Case Western Reserve University School of Graduate Studies / OhioLINK, 1996. http://rave.ohiolink.edu/etdc/view?acc_num=case1062085768.

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48

Tan, Yu Yin Nicole Medical Sciences Faculty of Medicine UNSW. "Gene expression during activation of smooth muscle cells." Publisher:University of New South Wales. Medical Sciences, 2009. http://handle.unsw.edu.au/1959.4/43615.

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Cardiovascular disease, which involves the cardiac, cerebrovascular and peripheral vascular system, is the major cause of morbidity and mortality in the western world. Changes in the vascular microenvironment trigger cascades of molecular events involving altered signaling, transcription and translation of a gene. The aim of this thesis was to increase our understanding on the molecular regulation of activated vascular smooth muscle cells. The first study looking at PDGF-D expression provides new insights into the regulatory mechanisms controlling the phosphorylation of Sp1. Studies performed identified three amino acids in Sp1 (Thr668, Ser670 and Thr681) that is phosphorylated by PKC-zeta activated by AngII. In the second study, the translational regulatory role of a novel gene YrdC induced by injury was investigated. Current knowledge of translational regulators controlling altered gene expression is little and studies in this thesis shows a splice variant of YrdC playing an important role in controlling mRNA translation and thus protein synthesis in the context of injury. The final study investigated in this study was the increased expression of the apoptotic FasL by the activation of GATA6. Although FasL has been extensively studied over the years, this is the first study linking a GATA factor with FasL in any cell type and provides key insights into the transcriptional events underpinning FasL-dependent SMC apoptosis following exposure to AngII.

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Oancea, Adriana Ecaterina. "Immunoglobulin heavy chain gene expression in hybridoma cells." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0010/NQ28030.pdf.

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50

Norgate, Zoe. "Gene expression in rat uterine natural killer cells." Thesis, University of Cambridge, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.614884.

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